Discovery of a Siderophore Export System Essential for Virulence of <em>Mycobacterium tuberculosis</em>

<div><p>Iron is an essential nutrient for most bacterial pathogens, but is restricted by the host immune system. <em>Mycobacterium tuberculosis</em> (<em>Mtb</em>) utilizes two classes of small molecules, mycobactins and carboxymycobactins, to capture iron from the human host. Here, we show that an <em>Mtb</em> mutant lacking the <em>mmpS4</em> and <em>mmpS5</em> genes did not grow under low iron conditions. A cytoplasmic iron reporter indicated that the double mutant experienced iron starvation even under high-iron conditions. Loss of <em>mmpS4</em> and <em>mmpS5</em> did not change uptake of carboxymycobactin by <em>Mtb</em>. Thin layer chromatography showed that the Δ<em>mmpS4/S5</em> mutant was strongly impaired in biosynthesis and secretion of siderophores. Pull-down experiments with purified proteins demonstrated that MmpS4 binds to a periplasmic loop of the associated transporter protein MmpL4. This interaction was corroborated by genetic experiments. While MmpS5 interacted only with MmpL5, MmpS4 interacted with both MmpL4 and MmpL5. These results identified MmpS4/MmpL4 and MmpS5/MmpL5 as siderophore export systems in <em>Mtb</em> and revealed that the MmpL proteins transport small molecules other than lipids. MmpS4 and MmpS5 resemble periplasmic adapter proteins of tripartite efflux pumps of Gram-negative bacteria, however, they are not only required for export but also for efficient siderophore synthesis. Membrane association of MbtG suggests a link between siderophore synthesis and transport. The structure of the soluble domain of MmpS4 (residues 52–140) was solved by NMR and indicates that mycobacterial MmpS proteins constitute a novel class of transport accessory proteins. The bacterial burden of the <em>mmpS4/S5</em> deletion mutant in mouse lungs was lower by 10,000-fold and none of the infected mice died within 180 days compared to wild-type <em>Mtb</em>. This is the strongest attenuation observed so far for <em>Mtb</em> mutants lacking genes involved in iron utilization. In conclusion, this study identified the first components of novel siderophore export systems which are essential for virulence of <em>Mtb</em>.</p> </div

Cell envelope stress in mycobacteria is regulated by the novel signal transduction ATPase IniR in response to trehalose

<div><p>The cell envelope of mycobacteria is a highly unique and complex structure that is functionally equivalent to that of Gram-negative bacteria to protect the bacterial cell. Defects in the integrity or assembly of this cell envelope must be sensed to allow the induction of stress response systems. The promoter that is specifically and most strongly induced upon exposure to ethambutol and isoniazid, first line drugs that affect cell envelope biogenesis, is the <i>iniBAC</i> promoter. In this study, we set out to identify the regulator of the <i>iniBAC</i> operon in <i>Mycobacterium marinum</i> using an unbiased transposon mutagenesis screen in a constitutively <i>iniBAC</i>-expressing mutant background. We obtained multiple mutants in the <i>mce1</i> locus as well as mutants in an uncharacterized putative transcriptional regulator (<i>MMAR_0612</i>). This latter gene was shown to function as the <i>iniBAC</i> regulator, as overexpression resulted in constitutive <i>iniBAC</i> induction, whereas a knockout mutant was unable to respond to the presence of ethambutol and isoniazid. Experiments with the <i>M</i>. <i>tuberculosis</i> homologue (<i>Rv0339c</i>) showed identical results. RNAseq experiments showed that this regulatory gene was exclusively involved in the regulation of the <i>iniBAC</i> operon. We therefore propose to name this dedicated regulator <b><i>ini</i></b><i>BAC</i> <b>R</b>egulator (IniR). IniR belongs to the family of signal transduction ATPases with numerous domains, including a putative sugar-binding domain. Upon testing different sugars, we identified trehalose as an activator and metabolic cue for <i>iniBAC</i> activation, which could also explain the effect of the <i>mce1</i> mutations. In conclusion, cell envelope stress in mycobacteria is regulated by IniR in a cascade that includes trehalose.</p></div

Immunoblot analysis of IniR_Mtb with α-Strep antibody.

<p><i>M</i>. <i>smegmatis</i> containing the construct pEXCF-<i>iniR</i>_<i>Mtb</i>-Strep vector was cultured and cultures were exposed for 18 hours to 10 ng/ml ATc/ Strep-tagged IniR_Mtb was isolated with StrepTactin beads. Purified protein from the elution fractions (elution 1–5, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007131#pgen.1007131.s006" target="_blank">S6 Fig</a>) was exposed to either trehalose, ATP, both or neither. Presence is indicated with (+), absence is indicated with (-). Indicated on the image of the Western Blot are monomeric IniR (~90kDa) and possible dimers and tetramers.</p

Effect of <i>mmpS4</i> and <i>mmpS5</i> on the survival of mice infected with <i>M. tuberculosis</i>.

<p>Survival of mice infected with wt <i>Mtb</i> H37Rv (ML617), Δ<i>mmpS4/S5</i> (ML618), Δ<i>mmpS4/S5</i> singly complemented with <i>mmpS5</i> (ML619), Δ<i>mmpS4/S5</i> singly complemented with <i>mmpS4</i> (ML620), or Δ<i>mmpS4/S5</i> fully complemented with <i>mmpS4</i> and <i>mmpS5</i> (ML624). Thirteen mice were infected with each strain. Mice were euthanized at day 169.</p

MmpS4 and MmpS5 are required for siderophore secretion in <i>M. tuberculosis</i>.

<p>TLC of cell-associated and secreted siderophores extracted from cultures of wt <i>Mtb</i> H37Rv parent strain ML617, Δ<i>mmpS4</i> single deletion mutant ML472, Δ<i>mmpS5</i> single deletion mutant ML405, <i>mmpS4/S5</i> double deletion mutant ML618, Δ<i>mmpS4/S5</i> double deletion mutant fully complemented with <i>mmpS4</i> and <i>mmpS5</i> ML624, and the siderophore biosynthetic mutant Δ<i>mbtD</i>::<i>hyg</i> ML1424. Cultures were labelled with 7-[¹⁴C]-salicylic acid, which was run on the TLC as a control alongside ⁵⁵Fe-loaded cMBT and mycobactin (MBT). Lanes containing cell-associated extracts were loaded with 5,000 cpm, while media extracts were loaded with 7,500 cpm.</p

MmpS4 and MmpS5 are not involved in iron sensing or uptake of siderophores.

<p><b>A.</b> GFP fluorescence was measured in wt <i>Mtb</i> mc²6230, Δ<i>mmpS4/S5</i>, and Δ<i>mbtD</i>::<i>loxP</i> strains containing a <i>gfp</i>-based iron-regulated reporter construct. Strains were grown in 7H9 media and fluorescence was measured two days after the addition of carboxymycobactin (cMBT) (black bars) or blank control (grey bars). Experiments were performed in triplicate and are shown with standard deviations. <b>B.</b> Uptake of ⁵⁵Fe loaded cMBT by <i>Mtb</i> Δ<i>mmpS4/S5</i> (black circles) and Δ<i>mmpS4/S5</i> Δ<i>mbtD::hyg</i> (white triangles). Assays were performed at 37°C using a final concentration of 0.25 µM cMBT, 0.45 µCi ⁵⁵Fe in triplicate. Standard deviations are shown.</p

MmpS4 or MmpS5 is required for growth of <i>M. tuberculosis</i> under iron-limited conditions.

<p><b>A.</b> Expression of MmpS4 and MmpS5 in <i>Mtb</i>. Whole cell lysates from wt <i>Mtb</i> mc²6230, Δ<i>mmpS4</i>, Δ<i>mmpS5</i>, Δ<i>mmpS4/S5</i>, Δ<i>mmpS4/S5+mmpS4/S5</i>, Δ<i>mbtD</i>::<i>hyg</i>, and Δ<i>mmpS4/S5/mbtD</i>::<i>hyg</i> were probed by Western blot by using rabbit polyclonal antibodies raised against MmpS4 and MmpS5. The cytoplasmic fructose 1,6-bisphosphatase GlpX was used as a loading control and detected using an anti-GlpX antiserum. <b>B, C.</b> Serial dilutions of log-phase cultures of <i>Mtb</i> mc²6230, Δ<i>mmpS4</i>, Δ<i>mmpS5</i>, Δ<i>mmpS4/S5</i>, Δ<i>mmpS4/S5</i> fully complemented with <i>mmpS4</i> and <i>mmpS5</i>, Δ<i>mbtD</i>::<i>hyg</i>, and Δ<i>mmpS4/S5</i>Δ<i>mbtD</i>::<i>hyg</i> were spotted on low iron glycerol-alanine-salts (GAS) plates (<b>B</b>), or on low iron GAS plates with 5 µM hemoglobin as an iron source (<b>C</b>).</p

ChIP sequencing shows a clear binding peak for IniR_Mtb upstream of the <i>iniBAC</i> operon.

<p>Chromatin immunoprecipitation (ChIP) sequencing was performed to identify binding events of IniR_Mtb-FLAG_. To do so <i>M</i>. <i>tuberculosis</i> containing a vector encoding the FLAG-tagged IniR_Mtb was induced with ATc and crosslinked after induction. After lysis, chromatin was sonicated to small fragments and immuno-precipitated with anti-FLAG antibody. And purified and sequenced. A clear binding peak of IniR_Mtb upstream of the <i>iniBAC</i> operon could be identified. The blue line indicates the (+) strand reads and the red line indicates the (-) strand reads. The number on the X-axis represent the nucleotide position in the H37Rv genome.</p

Overview of the three domains that can be found in <i>iniR</i>.

<p>Noted domains are the most significant hits using Phyre2 structure prediction [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007131#pgen.1007131.ref021" target="_blank">21</a>]. The number between brackets indicates the confidence with which the Phyre2 prediction was made. A combination of NCBI BLAST and the Phyre2 server was used to obtain domain predictions. Domain I (amino acid 24 to 247) is an AAA+ ATPase domain sharing 15% amino acid identity with sso_1545 in <i>S</i>. <i>solfataricus</i>. Domain II (470–731) is 16% identical to domain III of MalT in <i>E</i>. <i>coli</i> and the C-terminal domain (745–812) contains a DNA binding domain sharing 20% identity to <i>E</i>. <i>coli</i> SdiA. Displayed domains are proportional to actual domain size.</p

MmpS proteins interact with MmpL proteins.

<p><b>A.</b> Genetic interactions between MmpS4 and MmpS5 proteins and their cognate MmpL proteins. Percent of growth in iron-restricted medium (7H9 medium containing 50 µM 2,2′-dipyridyl) of triple mutants Δ<i>mmpS4/L4/S5</i> and Δ<i>mmpS4/S5/L5</i> strains and those strains complemented with <i>mmpS4</i> or <i>mmpS5</i> compared to growth in iron-rich media. <b>B.</b> Interaction of the C-terminal soluble domain of MmpS4 (residues 52–140) with the L1 loop of MmpL4 (residues 58–199) by an <i>in vitro</i> pull down assay.</p